Specimens from 3 patients affected by NPC disease (two of them were siblings) and 3 normal controls have been used in this study. Samples from a patient affected by Sandhoff disease were used as positive control for ganglioside accumulation assays.
All 3 NPC patients presented the classical biochemical phenotype characterized by massive lysosomal/late endosomes accumulation of unesterified cholesterol in cultured fibroblasts. The diagnosis was confirmed by sequencing both NPC1 and NPC2 genes. All patients had mutations in the NPC1 gene. The two affected siblings presented the c.3182T>C (P.I1061T)/c.3182T>C (p.I1061T) genotype, while the third unrelated NPC patient presented the c.2795+1G>C/c.3493G>A (p.V1165M) genotype. This study was approved by the ethical committee of the University Hospital “S. Maria della Misericordia” and written consent was obtained from all subjects.
Stem cell selection and culture
Stem cell enriched cultures were obtained, both from skin biopsies and from already established skin fibroblast cultures at early passages (P1, P2, P3), adapting the methods previously described [19, 20].
Briefly, skin biopsies were minced and digested with 0.04% of collagenase type I (Sigma-Aldrich) for 15 minutes at 37°C. Cell suspension was filtered through a 40 μm nylon membrane (Dako) and 4 × 104 cells/mm2 were plated onto fibronectin coated dishes (10 μg/100 mm plate, from Sigma-Aldrich). In alternative, 2 × 106 cells directly isolated from human skin biopsies or 1 × 106 cells obtained from confluent primary skin fibroblast cultures, were seeded onto 100 mm plates coated with fibronectin and expanded at least for three passages in a selective media composed of 60% Dulbecco’s Modified Eagle medium (DMEM)/40% MCDB-201 (Sigma-Aldrich) supplemented with 1 mg/ml Linoleic Acid-BSA (Sigma-Aldrich);10-9 M dexamethasone (Sigma-Aldrich); 10-4 M Ascorbic acid-2 phosphate (Sigma-Aldrich); 1X Insulin-transferrin-sodium selenite (Sigma-Aldrich); 2% fetal bovine serum (FBS), (STEMCELL Technologies), 10 ng/ml human PDGF-BB (Peprotech EC); 10 ng/ml human EGF (Peprotech EC). Medium was replaced every 4 days and cells were split when they reached 70/80% confluence.
Single cell cloning
Stem cells, at the third passage in culture (P3), obtained from healthy donors (n = 600 cells) and NPC patients (n = 600 cells) were individually deposited directly into fibronectin-coated wells of 96-well plates (Falcon, BD-Biosciences, Italy) with an automated cell sorter (FACSAria, BD), and cultured in expansion medium supplemented with 10% FBS [19, 20]. To determine sorting efficiency and to verify if any well was seeded with more than one cell, we utilized the Vybrant CFDA SE (CFSE) as a cell tracker (Molecular Probes, Invitrogen). Wells were examined twice a week in order to determine the fraction of cells able to give rise to proliferating clones.
Multilineage differentiation was evaluated as previously described [19, 20].
Muscle cell- and endothelial cell- differentiation was achieved plating 0.5 to 1 × 104/cm2 cells in expansion medium containing 5% FBS (Sigma-Aldrich), 10 ng/mL bFGF, 10 ng/mL VEGF, and 10 ng/mL IGF-1 (all from Peprotech EC), but not EGF. Cells were allowed to become confluent and cultured for up to 2 weeks with medium exchanges every 4 days.
Hepatocytic differentiation was induced growing cells for two weeks at high density (2 × 104/cm2) onto fibronectin coated coverslips in a medium containing 0.5% FBS, 10 ng/ml FGF-4 and 20 ng/ml HGF (both from Peprotech EC).
For neurogenic differentiation, stem cells obtained after 3 passages in selective medium, were seeded at a density of 8000 cells/cm2 into 96 multiwell plates (BD Biosciences) or on coverslips. The differentiation protocol was adapted from a method previously described [19, 20]. Briefly, cells were plated in medium containing DMEM-HG with 10% FBS (called N1 medium). After 24 hours the DMEM-HG was replaced with fresh medium supplemented with 1% of B27 (Invitrogen), 10 ng/ml EGF (Peprotech) and 20 ng/ml bFGF (Peprotech) (called N2 medium) for 5 days. Thereafter, cells were incubated for 24/48 hours in DMEM supplemented with 5 μg/ml insulin, 200 μM of indomethacin and 0.5 mM IBMX (all from Sigma-Aldrich) without FBS (called N3 medium).
Flow cytometry analysis
After at least 3 passages in selective medium, stem cells were detached and stained with the following primary conjugated antibodies: CD10, CD13, CD29, CD49a, CD49b, CD49d, CD90, CD73, CD44, CD45, HLA-DR, CD117, CD34, CD271 (BD Biosciences), CD105, CD66e, KDR (Serotech), CD133 (Miltenyi Biotec), CXCR4, (R&D), ABCG-2 (Chemicon International). The percentage of cells expressing all considered antigens was determined by flow cytometry analysis (CyAn, Beckman Coulter). Properly conjugated isotype matched antibodies were used as negative controls.
Filipin staining was performed using the method described by Blanchette-Mackie et al. . Briefly, cells grown on coverslips, were incubated in serum free medium for 24 hours and then treated for 24 hours with LDL enriched medium. Cells were rinsed with PBS and fixed with 3% paraformaldehyde. After washing them with PBS, the cells were incubated with 1.5 mg of glycine/ml PBS for 10 minutes, stained with filipin (0.05 mg/ml, in PBS 10% FCS) for 2 hours and examined using a Zeiss fluorescence microscope.
Periodic acid Schiff staining (PAS)
PAS was employed to detect glycogen accumulation. Slides were oxidized in 1% periodic acid for 5 minutes, rinsed three times in distilled water and treated with Schiff’s reagent for 25 minutes. After extensive washing, slides were stained with Mayer’s hematoxylin for 10 minutes.
Protein markers: Cells were grown on coverslips then fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized 10 minutes at room temperature with 0.1% Triton X-100 (Sigma-Aldrich) and stained overnight at 4°C to visualize stem cell markers: Oct4 (Abcam, Rabbit polyclonal, 1:150), Nanog (Abcam, Rabbit polyclonal, 1:200), Sox2 (Millipore, Mouse monoclonal, 1:200), Nestin (Millipore, 1:100); myocytes specific markers: smooth muscle actin (SMA) (Sigma, 1:50 mouse monoclonal) and α-sarcomeric actin (ASA) (Sigma, 1:100 mouse monoclonal); endothelial cell marker: CD31 (Dako, 1:50 mouse monoclonal); hepatic specific marker: cytokeratins 8, 18, and 19 (CK) (1:50 mouse monoclonal); or neural specific markers: tubulin beta 3 (COVANCE, 1:1000 mouse monoclonal), NeuN, (Millipore, 1:50 mouse monoclonal) and MAP2 (Millipore, 1:50 rabbit polyclonal). Secondary antibody staining was done with donkey anti-rabbit or donkey anti-mouse antibodies (Alexa-Fluor 555 or 488, Molecular Probes) at 1:600 dilutions. Images were obtained with a live cell imaging dedicated system consisting of a Leica DMI 6000B microscope connected to a Leica DFC350FX camera (Leica Microsystems); 10X (numerical aperture: 0.25), 40X oil immersion (numerical aperture: 1.25) and 63X oil immersion (numerical aperture: 1.40) objectives were employed.
analysis: The analysis of GM2 and GM3 gangliosides was performed as previously described . Cells were grown on coverslips then fixed in 4% paraformaldehyde for 45 minutes at room temperature. Cells were then incubated at room temperature for 1 hour in blocking buffer [PBS with 10% normal donkey serum (NDS) and 0.02% saponin (Sigma-Aldrich)] and stained overnight at 4°C with mouse anti-GM2 (1:20 in blocking buffer) or mouse anti-GM3 (1:20 in blocking buffer). Cells were then incubated with a TRITC conjugated donkey anti-mouse IgM [1:80 in PBS with 2% NDS and 0.02% saponin (Sigma-Aldrich)]. For colocalization studies, a monoclonal anti-LIMP-1 (Novus Biologicals, Littleton, USA) was used as a primary antibody, and an Alexa fluor-conjugated anti-mouse (Invitrogen, Carlsbad, CA, USA) as a secondary antibody.
In all cases, nuclei were stained by DAPI (Vector Laboratories, Inc) and Vectashield (Vector) was used as mounting medium. Epifluorescence and phase contrast images were obtained with a live cell imaging dedicated system consisting of a Leica DMI 6000B microscope connected to a Leica DFC350FX camera (Leica Microsystems); 10X (numerical aperture: 0.25), 40X oil immersion (numerical aperture: 1.25) and 63X oil immersion (numerical aperture: 1.40) objectives were employed. Adobe Photoshop software was utilized to compose and overlay the images, and adjust contrast (Adobe, USA). The counts of positive cells were done manually, considering, where possible, 100 positive events per sample.
Total RNA was extracted from both non-confluent cultures of undifferentiated and differentiated cells at P3 using the TRIzol Reagent (Invitrogen). After treatment with DNase I (Ambion), first strand cDNA synthesis was performed with 1 μg total RNA using random hexanucleotides and MMLV reverse transcriptase (Invitrogen). Primers were designed from available human sequences using the primer analysis software Primer3 (Additional file 1: Table S1). Quantitative RT-PCR was performed using Roche LightCycler 480 Real-Time PCR System and the LightCycler 480 SYBR Green I Master (Roche), following manufacturer’s instructions. GAPDH was used as internal control for normalization. LightCycler 480 Basic software (Roche) utilized the second derivative maximum method to identify the crossing point (Cp).
Evaluation of apoptosis
Apoptosis in differentiated cells at P3 was evaluated by staining of phosphatidylserine exposed on cell membranes with FITC labeled Annexin V, according to the manufacturer’s instructions (Sigma-Aldrich) and analyzed by flow cytometry using a FACScan (Becton Dickinson, Franklin Lakes, NJ, USA).
Morphological analysis data were collected by using the BD Pathway bioimaging platform. Differentiated cells were first immunostained for the neural marker MAP-2, then images were acquired on a BD Pathway 855 using a 20X objective (0.75 NA) in the form of 2 × 2 montage. The images were then analyzed using BD’s Neurite Outgrowth Algorithm that automatically measure parameters describing neurite outgrowth.
Statistical analysis was performed using Student’s t test or one-way ANOVA test, followed by Bonferroni post-test. The analyses were carried out using the software Prism, version 4.0c, GraphPad Software, San Diego, CA, USA; JMP7, SAS Institute Inc., Cary, NC, USA.
p<0.05 was considered statistically significant.